PROJECT SUMMARY Inter-organelle contact sites, which form dynamically between two different organelles and represent sites for metabolite transfer, have become increasingly appreciated as essential regulators of cellular homeostasis. Recently, our lab identified novel membrane contact sites between mitochondria and lysosomes in non-neuronal cells, which allow for bidirectional regulation of lysosomal and mitochondrial dynamics including mitochondrial fission and highlight a new pathway through which the two organelles can interact. Interestingly, both mitochondria and lysosomes are implicated in cellular calcium homeostasis, and dysfunction in both organelles has been linked to neurodegenerative disease. Calcium homeostasis is particularly important in neurons, where in addition to regulating functions such as ATP production and cellular signaling, calcium also modulates excitability and neurotransmitter release, suggesting that a more tightly-regulated mechanism of calcium transfer between organelles could be beneficial in neuronal cell types. Importantly, our preliminary data in non-neuronal cells suggest that activation of lysosomal calcium release increases mitochondrial calcium and that disruption of mitochondria-lysosome contact sites alters these calcium dynamics. Given these data, elucidating if and how mitochondria-lysosome contact sites transfer calcium in neurons will be critical for understanding how calcium dyshomeostasis may contribute to pathologic processes such as neurodegeneration. In this project, I propose to investigate the mechanisms and regulation of calcium dynamics at mitochondria-lysosome contact sites and their subsequent dysfunction in neurodegenerative disease using advanced microscopy techniques including super-resolution live cell microscopy and calcium imaging in long-term cultures of human induced pluripotent stem cell (iPSC)-derived neurons. In Aim 1, I will investigate the mechanisms of bidirectional calcium transfer at mitochondria-lysosome contact sites using human-derived cortical neurons. Additionally, as several lysosomal calcium transporters, including ATP13A2, have been implicated in neurodegenerative diseases, dysregulation of calcium dynamics between the two organelles may represent a potential pathway driving neurodegeneration. In Aim 2, I will investigate how disease-linked loss-of-function mutations in ATP13A2, which cause Kufor-Rakeb syndrome, an atypical form of parkinsonism with dementia, alter mitochondria-lysosome contact site dynamics and calcium homeostasis, and further contribute to downstream dysfunction in both organelles in patient-derived cortical and midbrain dopaminergic neurons. Together, the proposed research and training plan will offer important opportunities to not only acquire new experimental techniques in advanced imaging and human disease modeling to foster my development as a physician-scientist, but to also gain insight into the molecular mechanisms underlying neurodegeneration. A better understanding of these neuronal pathways will facilitate the identification of novel therapeutic targets, which may ultimately improve patient outcomes.